WISP1 and TLR4 Signaling in ventilator-induced lung injury (VILI)

描述(申请人提供)：尽管急性肺损伤预后的改善在很大程度上归功于肺保护性机械通气策略，但呼吸机诱导的肺损伤(VILI)仍然是危重患者发病率和死亡率的重要因素。利用一项无偏见的全基因组关联研究，我们(Li等，Am J Resp Cell Mol Biol 2012)在小鼠VILI中发现了WNT1诱导的信号通路蛋白1(WISP1)。此外，我们和其他人已经确认，通过TLR4的先天免疫信号在VILI的发病机制中起着关键作用，拉伸诱导的WISP1表达及其促炎作用是TLR4依赖的。因此，我们提出：特定目的1.确定机械拉伸与呼吸道上皮WISP1生物合成和VILI偶联的分子途径。我们将：a)使用药理学和遗传学方法来剖析非规范Wnt信号通路在周期拉伸培养的小鼠呼吸道上皮细胞WISP1生物合成中的作用；b)使用生化决定因素来关联HTV(12ml/kg×6h)后小鼠完整肺中的非规范途径；以及c)通过野生型WISP1-/-以及在气管内慢病毒传递HTV后肺泡巨噬细胞中WISP1的原位沉默(从而留下上皮源WISP1作为唯一来源)，确定上皮源性WISP1在VILI中的作用。具体目的2.为了确定WISP1在传递机械应激反应到导致VILI的先天免疫系统中的作用：我们将通过比较HTV对野生型、全身TLR4和髓系细胞特异性TLR4缺失小鼠的影响来确定巨噬细胞(和中性粒细胞)TLR4信号在VILI中的作用(Lyz-TLR4；Nace等人，2013年)。具体目的3.通过TLR4确定WISP1作为辅助分子将机械牵张应激传递给巨噬细胞促炎表型的分子决定因素。我们将使用腹膜巨噬细胞定义WISP1的促炎作用(如肿瘤坏死因子α；核因子κB)的必要成分，并确认来自野生型和CD14CD3和ç5缺失小鼠的原代小鼠肺泡巨噬细胞培养(数量有限)中积累的信息。然后，我们将通过对比HTV在有和没有I.T.的情况下对B_3和B_5缺失小鼠的影响，来确定B_3和B_5在WISP1介导的VILI中的强制性作用。注射WISP1。综上所述，这些研究将对基质细胞蛋白WISP1和Wnt通路在机械应激对呼吸道上皮细胞的影响转导到天然免疫系统中的作用以及肺泡巨噬细胞TLR4激活在VILI发病机制中的作用提供新的见解。这种机制的洞察可能导致生物标志物、预防或缓解VILI的治疗靶点以及对VILI易感性的遗传决定因素的进一步了解。

DESCRIPTION (provided by applicant): Although much of the improvement in outcomes from acute lung injury has been ascribed to lung-protective mechanical ventilation strategies, ventilator-induced lung injury (VILI) remains an important element of morbidity and mortality in the critically ill patient. Using an unbiased genome-wide association study, we (Li et al, Am J Resp Cell Mol Biol 2012) identified a WNT1-inducible signaling pathway protein 1 (WISP1) in murine VILI. Moreover, we and others have identified that innate immune signaling via TLR4 plays a critical role in the pathogenesis of VILI and that stretch-induced WISP1 expression and its pro-inflammatory effect were TLR4- dependent. Accordingly, we propose: Specific Aim 1. To determine the molecular pathway by which mechanical stretch is coupled to respiratory epithelial WISP1 biosynthesis and VILI. We will: a) use pharmacological and genetic approaches to dissect contribution of non-canonical Wnt signaling pathway in WISP1 biosynthesis in cyclic stretched cultured murine respiratory epithelium; b) use biochemical determinants to associate non-canonical pathway in intact lung of mice after HTV (12 ml/kg x 6h); and c) determine the contribution of epithelial derived WISP1 in VILI by changes in alveolar capillary permeability after HTV in wildtype, WISP1 -/- and after silencing WISP1 in alveolar macrophages in situ with intratracheal lentiviral delivery of shRNA (and hence leaving epithelial derived WISP1 as sole source). Specific Aim 2. To determine the role of WISP1 in communicating mechanical stress responses to innate immune system leading to VILI: We will determine the contribution of macrophage (and neutrophil) TLR4 signaling in VILI by comparing the effect of HTV on wildtype, whole body TLR4 and myeloid-cell-specific TLR4 null mice (Lyz-TLR4; Nace et al, Hepatology 2013). Specific Aim 3. To determine the molecular determinants by which WISP1 acts as an accessory molecule transducing stress of mechanical stretch in airway epithelium to pro-inflammatory phenotype of macrophages via TLR4. We will define requisite components for proinflammatory (e.g. TNFα; NFκB) effect of WISP1 using peritoneal macrophages and confirm accrued information in primary murine alveolar macrophage cultures (that are limiting in number) from wildtype and CD14 and ß3 and ß5 null mice. We will then determine the obligatory roles of ß3 and ß5 in WISP1 mediated VILI by contrasting the effect of HTV on ß3 and ß5 null mice, with and without i.t. injection of WISP1. Collectively, these studies will provide novel insight into the role of matricellular protein, WISP1, and the Wnt pathway, in transducing the effect of mechanical stress on respiratory epithelium to innate immune system and role of TLR4 activation of alveolar macrophages in pathogenesis of VILI. Such mechanistic insight may lead to biomarkers, therapeutic targets in prevention or mitigation of VILI and further understanding of genetic determinants of susceptibility to VILI.